1、掀起掀起PolyplusPolyplus转染风暴转染风暴产品专员培训产品专员培训JPolyplus-transfectionPolyplus-transfection转染方法介绍转染方法介绍：显微注射法电穿孔法 (Amaxa)Biolistic 颗粒传递法 （基因枪粒子轰击法）磷酸钙法阳离子脂质体法（Invitrogen，Roche，Qiagen ）阳离子聚合物 (Sunma梭华，凯基，polyplus)物理物理方法方法化学化学方法方法病毒介导法病毒介导法 转染的应用：转染的应用： 基因组功能研究：基因表达调控，蛋白功能，信号转导和药物筛选研究 建立细胞系：转基因细胞模型、永生性细胞株 建立转
2、基因动物模型 基因治疗研究和应用 蛋白功能和定位研究 重组蛋白生产及蛋白药物研究Why do we need transfection reagent?DNA: highly anionic and hydrophilic macromoleculeNo ionic interaction+Cationic macromolecule+Cationic ReagentMembrane: anionic and hydrophobic-Heparan Sulfate Proteoglycanes细胞转染难易比较Adherent Suspension Problem:E.g.:MembraneTH
3、P1, JurkatFast dividing cell non dividing cellProblem:E.g.:Access to the nucleusNeurons,Cell line Primary cellProblem:E.g.:More sensitiveEpithelial primary cellsv PolyPlus-transfection PolyPlus-transfection是法国一家专门专门致力于研发和销售转染试剂的生物技术公司PolyPlus-transfection公司简介公司简介v PolyPlus-transfectionPolyPlus-trans
4、fection提供体内体内和体外体外转染产品，包括基因、寡核苷酸、 siRNA及蛋白转染产品，可进行瞬时瞬时和稳定稳定的转染及重组蛋白质制备。第一部分第一部分 产品介绍产品介绍In VitroIn VivosiRNA transfection Standard transfectionINTERFERinjetSI-ENDOTrafficking and visualizationjetSI-FluojetSI-ENDO-Fluo产品产品介绍介绍DNA transfection Standard transfection in vivo-jetPEILigand-conjugated reag
5、entsin vivo-jetPEI-Gal in vivo-jetPEI-ManTrafficking and visualization in vivo-jetPEI-Fluoin vivo-jetPEI-biotin DNA transfection In vitroIn vivoStandard transfection jetPEI Cell-specific transfectionjetPEI-GaljetPEI-MacrophagejetPEI-HUVECjetPEI-RGD Trafficking and visualization jetPEI-FluojetPEI-bio
6、tin Synthetic medium transfectionFecturinProtein deliveryProtein and antibody PULSinin vivo-jetPEIMouse brain injectionjetSI 10 mMsiRNA transfection 产品系列产品系列 Protein&antibody 转染转染 - PULSinSiRNA 体外转染体外转染 - INTERFERin DNA&SiRNA 体内转染体内转染 - in vivo jetPEIDNA 体外转染体外转染 - in vitro jetPEI产品系列一产品系列一Protein &
7、 antibody 转染: -PULSin蛋白蛋白Protein&antibody转染应用：应用：细胞内定位研究蛋白水平的干扰研究抗体功能封闭的研究疾病治疗的研究替代免疫细胞化学在活细胞内检测蛋白蛋白protein 转染难题 Mass Charge Purity Stability 3D structure Release Activityx kDapI = xx %hours or daysGlobular, random, well-defined structureFree protein in cytoplasmDNA and RNA = anionic polymers Protei
8、n = more complex molecules 蛋白蛋白技术原理技术原理PULSin: cationic amphiphilic-based formulation蛋白蛋白操作步骤操作步骤21For 24-well plate:细胞融合度：70-80%稀释蛋白：1g of protein/100l Hepes1加 4 l PULSin2孵育 15 min3洗细胞，加900 l无血清培养基4 加protein/PULSin复合物345孵育 4 h5更换新鲜完全培养基6 分析蛋白活性或检测6蛋白蛋白R-phycoerythrinNIH-3T3 cellsConditions: Living
9、cells were analyzed by fluorescence microscopy 16 hours post-delivery.pI = 5.5MW = 240 kDaPULSinPULSin转染蛋白质转染蛋白质1 g R-PE+ - 4 l PULSin+- - Diffuse fluorescence Efficient delivery of protein with PULSin蛋白蛋白PULSinPULSin转染单抗转染单抗HeLa cellsanti-NPC-(AF488)1 l Ab4 l PULSinConditions: Living cells were ana
10、lyzed by fluorescence microscopy 8 hours post-delivery. Localized fluorescence around nuclear membrane Efficient delivery of functional Ab抗体抗体PULSinPULSin转染转染多抗多抗HeLa cellsanti-giantin-(AF488)0.5 l Ab4 l PULSinConditions: Living cells were analyzed by confocal microscopy 17 hours post-delivery. Plas
11、ma membranes were stained with ConcanavalinA-rhodamine. Efficient delivery of functional Ab抗体抗体PULSinPULSin转染转染多肽多肽多肽多肽HeLa cellsConditions: Delivery of Pep-A (Streptococcus TPE B epitope,), into HeLa cells. Complexes were formed with Pep-A (1 g, lissamine-rhodamine derivative, Sigma) and PULSin (4
12、l). Observation was carried out 16 h post-delivery. Pep-A (Streptococcus TPE B epitope)16 aa， lissamine-rhodamine derivative Efficient delivery of PeptidesPULSinPULSin已已转染的不同细胞类型转染的不同细胞类型R-PE: 1 g / PULSin: 4 l Delivery to a wide variety of cells蛋白蛋白PULSinPULSin已转染的细胞及转染效率已转染的细胞及转染效率Adherent cell li
13、nes3T3 L1A549BHK-21CaSkiCHOCV-1HEK-29360-80%80%30-40%80-90%80-90%50%45-55%HeLa HepaRGMCF-7MLE-15 NIH-3T3RAW 264.7Si Ha 80-90%60-70 %60%60-75%90-98%40-50%60-70%Suspension cell linesJurkat THP-120-30%10%K-56220-30%Adherent primary cellsPrimary human fibroblastsPrimary human hepatocytesPrimary human ke
14、ratinocytesPrimary human visceral preadipocytes60-70%50-40%55-70%60-75%蛋白蛋白R-phycoerythrinR-phycoerythrinR-phycoerythrinProteinsR-phycoerythrin -galactosidaseBovine Serum Albumin (FITC) Histone H1AntibodiesAnti- -tubulin (FITC, mouse IgG1) Anti-vimentin (FITC, mouse IgG2a)Anti- -actin (PE, mouse IgG
15、2a) Anti- -actin (FITC, mouse IgG1)Anti-giantin (AF488, rabbit polyclonal)Anti-Nuclear Pore Complex Proteins (AF488, mouse IgG1)Goat anti-mouse IgG (FITC) Goat anti-mouse IgG (PE)PULSin: PULSin: 蛋白蛋白/ /抗体抗体/ /多肽列表多肽列表蛋白蛋白多肽多肽大小大小pIpI疏水疏水aaaa的的带电带电aaaa的数量的数量标记标记9-mer3.378%1-Lissamine rhodamine9-mer6.
16、044%0Lissamine rhodamine10-mer3.030%4-Lissamine rhodamine12-mer7.842%1+FITC16-mer2.831%5-Lissamine rhodamine优势：优势： 新新技术活细胞中进行Protein/Ab转染 高转染效率 无细胞毒性 即用型溶液 操作简单PULSinPULSin优势优势蛋白蛋白PULSinPULSin的竞争对手的竞争对手蛋白蛋白Chariot (Active Motif)BioPorter (GTS)ProteoJuice (Merck)产品系列二产品系列二体外 SiRNA 转染: -INTERFERin选择、设
17、计、合成siRNA双链化学合成siRNA质粒表达siRNA 转染siRNA到靶细胞瞬时表达 化学合成siRNA，质粒表达siRNA稳定表达 质粒表达siRNA 分析检测RNA干扰作用蛋白水平: WB/ IF/ FCmRNA水平: 实时定量RT-PCR，northern blottingsiRNA基因沉默实验设计基因沉默实验设计iRNA介导基因沉默机制介导基因沉默机制 siRNAsiRNA expressionplasmid Cleavage byDicer siRNAssiRNA associate with RISC Cleavage of mRNA by RISC RISCdsRNADic
18、er cleavage of dsRNAHairpin siRNA Cell membraneProtein XINTERFERinjetPEIsiRNA 实验操作实验操作 siRNA (1 nM) in serum-free medium1Add INTERFERinVortex and incubate 10 min at r.t.31234Add to well containing cells in complete mediumIncubate for 48 hours at 37C56Gene silencing assay6542Protocol for cells in 24-
19、well plateINTERFERinsiRNA 反向操作反向操作HTS操作操作 Standart protocolReverse protocolPlate cells 24h prior to transfectionDilute siRNAsAdd INTERFERinVortex Incubate 10 minDistribute into wells Distribute siRNAs or use librariesAdd INTERFERinMixIncubate 10 minAdd cells96-well plateDay 1Day 2Day 1Day 3PlateTran
20、sfectAssayPlate and transfectDay 4AssaysiRNA 反向转染基因沉默效率反向转染基因沉默效率 A549-GL3Luc Luciferase96-well plate反向转染仍能得到极高的基因沉默效率siRNA siRNA delivery with INTERFERin CaSki cells (24-well plate) Lamin A/C Immunofluorescence assay1 nM siRNA siRNA 基因沉默效率基因沉默效率基因沉默效率基因沉默效率Great silencing down to picomolar siRNA co
21、ncentration A549-GL3Luc cells (24-well plate) Luciferase70 %92 %siRNA 细胞毒性细胞毒性 A549-GL3Luc (24-well plate)1 nM siRNA No cellular toxicity observed with INTERFERinL1INTERFERinL2siRNA 成功转染的细胞系成功转染的细胞系Silencing efficiency with INTERFERin in various cell lines贴壁细胞 (at 1 nM siRNA conc.)A549Luciferase90,9
22、%HeLaGAPDH95.7%CaskiGAPDH94.7%SiHaGAPDH92.5%MCF7GAPDH90 %NIH-3T3Vimentin89.8%悬浮细胞 (at 5 nM siRNA conc.)K562GAPDH78.8%THP-1GAPDH81.8%siRNA 原代细胞的高效沉默原代细胞的高效沉默 24-well plate GAPDH(甘油醛-3-磷酸脱氢酶) Branched DNA assay96 %人原代肝实质细胞人原代纤维原细胞82 %siRNA INTERFERinINTERFERin优势优势 低浓度低浓度 siRNA（甚至甚至pmol级别）就能达到高效的级别）就能达
23、到高效的基因沉默基因沉默 在很多细胞系中，基因沉默效率可达到在很多细胞系中，基因沉默效率可达到90以上以上 在原代细胞中也能达到有效的基因沉默效率在原代细胞中也能达到有效的基因沉默效率 无细胞毒性无细胞毒性 不受血清和抗生素的影响不受血清和抗生素的影响 操作简单操作简单 价格便宜价格便宜 在反向操作转染中的效率高在反向操作转染中的效率高siRNA siRNA siRNA 转染竞争对手转染竞争对手Lipofectamine 2000 （ Invitrogen）Oligofectamine （ Invitrogen）HiPerFect （Qiagen）SiLentFect （BioRad） Inv
24、itrogen is the largest siRNA transfection market Qiagen is a dangerous outsider with HiPerFectsiRNA INTERFERin vs Lipofectamine 2000INTERFERinLipofectamine 20001. siRNA 浓度浓度1 nM siRNA（降低成本）20-100nM siRNA(40nM)2. 血清和抗生素血清和抗生素不受血清和抗生素影响不能加入抗生素3. 基因沉默效率基因沉默效率90以上?4. 操作操作即用型，简单需稀释，复杂5. 细胞毒性细胞毒性无细胞毒性细胞毒性
25、大6. siRNA/reagent (/well in 24well)0.6pmol/1-3ul10-50pmol/0.5-1.5ul7. 价格价格INTERFERin vs OligofectamineINTERFERinOligofectamine1. siRNA 浓度浓度1 nM siRNA100nM siRNA2. 血清和抗生素血清和抗生素不受血清和抗生素影响不能加入抗生素3. 基因沉默效率基因沉默效率90以上一般4. 操作操作即用型，简单需稀释，复杂5. 细胞毒性细胞毒性无细胞毒性细胞毒性大6. siRNA/reagent (/well in 24well)0.6pmol/1-3ul
26、60pmol/3ul7. 价格价格INTERFERin vs HiPerFect/ SiLentFectINTERFERinHiPerFectSiLentFect1. siRNA 浓度浓度1 nM siRNA5nM siRNA10nM siRNA2. 细胞融合度细胞融合度30-5050-8050-903. 基因沉默效率基因沉默效率高，见表高，见表见表见表见表见表4. 细胞毒性细胞毒性无细胞毒性无细胞毒性细胞毒性较小细胞毒性较小细胞毒性大细胞毒性大5. siRNA/reagent (/well in 24well)0.6pmol(8.4ng)/1-3ul37.5ng/3ul6. 价格价格INTE
27、RFERin与HiPerFect/ SiLentFect基因沉默效率基因沉默效率 A549-GL3Luc (24-well plate) LuciferaseINTERFERin与HiPerFect/ SiLentFect细胞毒性细胞毒性 A549-GL3Luc (24-well plate)1 nM siRNA No cellular toxicity observed with INTERFERinHiPerFectINTERFERinSiLentFect体内 DNA&siRNA转染: -in vivo jetPEI产品系列三体内体内 in vivo jetPEI vs Virusesin
28、 vivo jetPEI :easy to be usednot immunogenicnot toxic can delivered oligonucleotides up to very large DNA but also siRNA体内体内 Efficiency/specificity+Virusin vivo-jetPEIgene size(kbp)3 - 30 400YesnoneImmune responseYes (L2 L3)noneLab requirement体内体内 in vivo jetPEI vs VirusesDifferent routesVarious ani
29、mals Intravenous静脉 Intratumoral瘤内 Intracerebral 脑室 Intraperitonal腹膜 Intratracheal气管灌注 Instillation 吸入 Mouse Rat Duck Guinea pig Monkey 体内转染的路径和动物 towards Human application体内体内 体内转染的基因表达分布情况O. Freund 活小鼠体内荧光素酶表达活小鼠体内荧光素酶表达的荧光检测图象的荧光检测图象（用 含50 g pCMVluc 和 in vivo-jetPEI 复合物的400 l 5% 葡萄糖溶液尾部注射Balb/C mi
30、ce ） Liver 30 000Tumor 2 000Kidney L300 000Lung R20 000 000Kidney R115 000Ovary L210 000 Lung L16 000 000Ovary R150 000 Courtesy JL. Coll and O. Freund in vivo-jetPEI体内转染体内体内 In RatsH19-DTA（H19-白喉毒素A基因）质粒与 in vivo-jetPEI复合物转入大鼠膀胱。NBT-II cells50g DNA at D+4 & D+8D+11 rats were sacrifiedOhana, P., et
31、al. Gene Ther Mol Biol (2004) 8, 181-192Decrease in bladder weightInhibition of tumor growthin vivo-jetPEI转染对膀胱癌的治疗体内体内 In HumanH19-DTA plasmid /in vivo-jetPEI复合物转染对人膀胱癌的治疗Ohana, P., et al. Gene Ther Mol Biol (2004) 8, 181-192(D) Video-cystoscopy performed before the treatment and three weeks after
32、the completion of the 6th treatment, showing a large necrotic area replacing the tumor region (E).Treatment shows 75% tumor regressionin vivo-jetPEI转染对膀胱癌的治疗体内体内 活体小鼠尾静脉注射（活体小鼠尾静脉注射（24小时后的荧光检测图）小时后的荧光检测图）24 hours later, the luciferase gene expression was monitored by bioluminescent imaging of live m
33、ice using a cooled CCD camera. DNA alone (GL2-Luc)DNA (GL2-Luc) +GL2-Luc-siRNA, 10 gDNA (GL2-Luc) +GL3-Luc-siRNA, 10 gColl, Freund, Erbacherin vivo-jetPEI : DNA-siRNA共转染 siRNA and pCMVLuc 与 in vivo-jetPEI复合物转染对荧光素酶肺部表达起明显的抑制作用。体内体内 Urban-klein et al., Gene Therapy, 1-6, 2004C-erb2/neu (HER-2) recept
34、orin vivo-jetPEI：-体内基因沉默HER-2 down regulationTumor growth inhibition during 18 days i.p. in athymic nude miceSubcutaneous tumor xenografts SKOV-3 (ovarian carcinoma cell)A) Naked HER-2 specific siRNA (0.6 nmole)B) HER-2 siRNA/PEI polyplexes体内体内 产品系列四产品系列四DNA in vitro transfection: - in vitro jetPEID
35、NA转染过程1. 提取质粒DNA或寡核苷酸2. 选择和培养细胞系3. 转染转染瞬时转染稳定转染：抗性筛选4. 检测外源基因细胞内表达的实验流程：外源基因细胞内表达的实验流程：DNA转染试剂+H3NO+ONH3CCH3H3CDOTMA (Lipofectin) DOTMA (Lipofectin) Monocationic lipidsPolycationic lipids+NONOHNHNH3H2NTransfectam Transfectam Cationic polymersPAMAM Dendrimer PAMAM Dendrimer OHONHONHOH2NNHRNHRNHRnPoly
36、lysine and derivativesPolylysine and derivativesNNNNNNNNNNH2NNH2N H2NH2NH2N H2NH2H2NH2NH2NH2NN H2012GenerationsNHNNHNHNNHNNHNHNHNNH3+NHNHNNHNHNNNHNH2NH+3HN+3HNNH3+NH3+NH3+Polyethylenimine(PEI) Polyethylenimine(PEI) polyethylenimine (PEI)NH= -CH2-CH2-NH- = 43 Daammonium groupsfor DNA condensationbuff
37、eringamine groupsbranched PEIlinear PEI1H+/amine3579pH0.5nnjetPEIDNAjetPEICationic complexesnucleusProton SpongecytoplasmEndocytosisin vitro DNA deliveryNHHNOHnmHCl-H3CjetPEI 1 l = 7.5 nmoles of amine functionDNA 1 g = 3 nmoles of phosphate groupnumber of nitrogen residues of jetPEInumber of DNA pho
38、sphates=NP Complexes Formation2 l of jetPEI for 1 g of DNA at N/P = 5 jetPEI: 实验操作+ serum2 l of jetPEI for 1 g of DNA1 ml of jetPEI = 500 transfections in 24-well platesor = 2500 transfections in 96-well platesHeLa cells1 g of pCMVLuc, N/P 5Complexes FormationEffect of incubation timeSame efficiency
39、HeLaHEK293NIH 3T3-galactosidase BHKN/P = 3N/P = 5controlpCMVLacZReproductibilityCells transfected in 24-well plates with jetPEI ( N/P = 5) with a -galactosidase-expressing plasmid (pCMVLacZ).Expression was visualized by X-gal staining after 24 h. 60 80 %60 80 %40 60 %jetPEI: 转染效率细胞特异性jetPEI转染产品jetPE
40、I-Macrophage（巨噬细胞）jetPEI-Mannose / Mannose receptor / MacrophagesjetPEI-Hepatocyte（肝实质细胞）jetPEI-Galactose / Galactose receptor / HepatocytesjetPEI-HUVEC（人脐静脉内皮细胞）jetPEI-Glucose / HUVEC cellsjetPEI-Fluo & BiotineTransfection positive control 价格优势价格优势（低10%-20%） 实验操作简易实验操作简易 与血清和抗生素兼容性好与血清和抗生素兼容性好 稳定性好
41、稳定性好(质子海绵假说） 、毒性低毒性低 规格齐全，能满足不同客户需求规格齐全，能满足不同客户需求 保质期长达一年保质期长达一年jetPEI优势jetPEI DNAjetPEI DNA转染竞争对手转染竞争对手Lipofectamine 2000 (Invitrogen)Fugene 6 (Roche)Effectene (Qiagen)CaPO4 Invitrogen, Roche & Qiagen70% transfection marketjetPEI vs Lipofectamine 2000jetPEILipofectamine 20001.试剂成分试剂成分线性阳离子聚合物线性阳离子聚
42、合物阳离子脂质体阳离子脂质体2. 细胞融合度细胞融合度50-60 90-953. 血清和抗生素血清和抗生素不受血清和抗生素影响不受血清和抗生素影响不能加入抗生素不能加入抗生素4. 转染效率转染效率高高高高5. 细胞毒性细胞毒性无细胞毒性无细胞毒性细胞毒性大细胞毒性大6. DNA/reagent(/well in 24well)1ug/2ul0.8ug/2ul(1:2-1:3)7. 价格价格jetPEI vs Fugene 6(Roche)jetPEIFugene 61. 成分成分线性阳离子聚合物线性阳离子聚合物脂质聚合体和特定成分组成脂质聚合体和特定成分组成2. 细胞融合度细胞融合度50-60
43、50-803. 血清和抗生素血清和抗生素不受血清和抗生素影响不受血清和抗生素影响不能加入抗生素不能加入抗生素4. 转染效率转染效率高高高高5. 细胞毒性细胞毒性无细胞毒性无细胞毒性细胞毒性大细胞毒性大6. DNA/reagent(/well in 24well)1ug/2ul0.2ug/0.6ul(1:6,1:3,2:3)*7. 价格价格jetPEI vs Effectene(Qiagen)jetPEIEffectene1. 成分成分线性阳离子聚合物线性阳离子聚合物非脂质体脂质类非脂质体脂质类2. 细胞融合度细胞融合度50-6040-803. 血清和抗生素血清和抗生素不受血清和抗生素影响不受血
44、清和抗生素影响不受血清和抗生素影响不受血清和抗生素影响*4. 转染效率转染效率高高?5. 细胞毒性细胞毒性无细胞毒性无细胞毒性细胞毒性小？细胞毒性小？6. DNA/reagent(/well in 24well)1ug/2ul0.2ug/5ul7. 价格价格jetPEI vs CaPO4 jetPEI is A POLYMER more efficient a better reproducibility easy protocol第二部分第二部分产品推广产品推广推广策略v 试用装策略试用装策略v 全国技术讲座策略全国技术讲座策略 v 转染小册子策略转染小册子策略 v 重点客户策略重点客户策略
45、v 新产品为切入点策略新产品为切入点策略v 其他资料：成功转染细胞系、发表文献其他资料：成功转染细胞系、发表文献v 全程技术服务全程技术服务一、寻找客户实验室：实验室： 分子生物学实验室分子生物学实验室 细胞生物学实验室细胞生物学实验室 基因研究实验室基因研究实验室 动物研究实验室动物研究实验室 蛋白组学研究实验室蛋白组学研究实验室二、了解客户了解客户实验情况：了解客户实验情况： 正在使用的转染试剂：脂质类， CaPO4, 电转染, virus 转染DNA、siRNA、蛋白、寡核苷酸？体内、体外？ 转染的细胞系？ 瞬时转染或稳定转染？ 转染的效率或基因沉默的效率？满意吗？ 检测方法？二、了解客户 估计客户或实验室用量：估计客户或实验室用量： 询问实验室其他人的实验情况：询问实验室其他人的实验情况：三、总结 -总结客户的资料，推荐相应的产品，突出我们产品的优势。 - 计划下一客户的拜访 总结和计划总结和计划
